Abstract The cardiac myocyte has long been the primary focus of studies attempting to elucidate the regulatory aspects underlying cardiac development and disease. However, recently the involvement of nonmyocytes has emerged as potentially just as important as myocytes in contributing to and controlling cardiac remodeling during development and progressive pathogenesis in ischemia-induced heart failure. More specifically, the cardiac fibroblast and its ability to convert to myofibroblasts in promoting ECM production, ventricular remodeling and the fibrotic response have been underappreciated as critical regulator of cardiac biology. Here we propose a dual-PI application led by developmental and adult disease-based cardiac investigators to address key areas of fibroblast biology that span the early postnatal heart up through the failing and fibrotic adult heart. Previously, the field had not been able to carefully annotate the functional aspects of the fibroblast within the developing and diseased heart, in part because of a lack of appropriate genetic tools that specifically target this cell-type. More recently we and others have generated a few critical genetically modified mouse models that specifically target resident cardiac fibroblasts, as well as all activated fibroblasts and myofibroblasts in the heart. Thus, here we can now test the novel and overarching hypothesis that activated fibroblasts and myofibroblasts play selective roles in early neonatal ventricular maturation, regeneration and heart development, which is recapitulated in the adult heart in response acute and chronic ischemia-induced disease states. The dual-PI application has 3 specific aims: 1) To define sub-stages and functions of cardiac fibroblasts and myofibroblasts during postnatal development and in the adult heart following acute and chronic disease stimulation, 2) To identify crosstalk mechanisms between cardiac fibroblasts and myocytes in the developing and diseased heart, and 3) To define Tcf21- mediated contributions to fibroblast lineage expansion and commitment in the developing postnatal heart and in the adult heart after ischemic and acute injury. Collectively, these specific aims will uncover stages of fibroblast differentiation during development, regeneration and hypertrophy across both models of interstitial fibrosis and replacement fibrosis. Such an understanding will lay the foundation for future studies into specific therapeutic pathways to target in treating longstanding fibrotic heart disease states or to enhance the regenerative capacity of the heart.